A Method for Intracellular Autoradiography in the Electron Microscope

A technic is described for high resolution intracellular autoradiography in the electron microscope. Cultures of LLC-MK(2) monkey kidney cells were incubated for 72 hours in a medium containing 0.4 microcurie per ml of thymidine-H(3). After labeling, the cells were fixed with osmium tetroxide and embedded in methacrylate. Ultrathin sections of the labeled tissue were taken up on Formvar-coated and carbon-stabilized electron microscope grids. A 150 to 450 A layer of silver metal was then evaporated onto the tissue. The coated grids were exposed to bromine vapor for 1.5 to 2 minutes under red light, allowed to dry for 1 minute, and then covered with a thin film of 1 per cent aqueous gelatin applied by means of a fine wire loop lowered over the grid supported on a glass peg. For autoradiographic exposure, the grids were stored 50 days in a light-proof container at 4 degrees C with calcium chloride desiccant. Development was carried out for 5 minutes at 20 degrees C in Promicrol (May and Baker, England) diluted 1:1 with water, followed by a 1 minute water wash and fixation for 2.5 minutes in 15 per cent aqueous sodium thiosulphate. After removal of the gelatin by immersion for 16 hours in water at 37 degrees C, the autoradiograms were dried and examined in the electron microscope. Ultrastructural detail was fairly well defined and the cytoplasm of each labeled cell was covered with an electron opaque deposit of silver, suggesting that a polynucleotide containing thymidine may be synthesized in the cytoplasm. The matter is discussed.